The Cyclin E1 gene (CCNE1) is an ideal model to explore the mechanisms that control the transcription of cell cycle-regulated genes whose expression rises transiently before entry into S phase. E2F-dependent regulation of the CCNE1 promoter was shown to correlate with changes in the level of H3-K9 acetylation͞methyl-ation of nucleosomal histones positioned at the transcriptional start site region. Here we show that, upon growth stimulation, the same region is subject to variations of H3-R17 and H3-R26 methylation that correlate with the recruitment of coactivator-associated arginine methyltransferase 1 (CARM1) onto the CCNE1 and DHFR promoters. Accordingly, CARM1-deficient cells lack these modifications and present lowered levels and altered kinetics of CCNE1 and DHFR mRNA expression. Consistently, reporter gene assays demonstrate that CARM1 functions as a transcriptional coactivator for their E2F1͞DP1-stimulated expression. CARM1 recruitment at the CCNE1 gene requires activator E2Fs and ACTR, a member of the p160 coactivator family that is frequently overexpressed in human breast cancer. Finally, we show that grade-3 breast tumors present coelevated mRNA levels of ACTR and CARM1, along with their transcriptional target CCNE1. All together, our results indicate that CARM1 is an important regulator of the CCNE1 gene.ACTR ͉ CCNE1 ͉ histone ͉ arginine methylation ͉ breast tumor C yclin E1 (CCNE1) protein and mRNA levels are tightly regulated as an endpoint of several regulatory pathways that are critical for growth control and frequently altered in cancer cells (1, 2). CCNE1 gene transcription is undetectable in G 0 and G 1 phases of the cell cycle, whereas it rises sharply during a narrow window of time that precedes each entry into S phase. Several pieces of evidence suggest that the periodic association of activators E2Fs-and E2F-pocket protein complexes regulate CCNE1 gene expression (3-18). E2F complexes bound to this gene were found to recruit chromatin modifiers, including members of the SNF2-like helicase family, type I histone deacetylases, the acetyltransferase CBP͞p300, the lysine methyl transferase SUVAR39H1, and the protein arginine N-methyltransferase (PRMT) 5 (7, 9-14, 17, 18), suggesting that they foster periodic chromatin remodeling of the CCNE1 promoter region (11,12,14). Notably, repression of the CCNE1 gene in G 0 -G 1 correlates with the methylation of H3-K9 and H4-R3 on a single nucleosome positioned at the transcriptional start site (11)(12)(13)(14). Conversely, the late G 1 activation of the CCNE1 gene correlates with decreased H3-K9 methylation and with enhanced H3͞H4 acetylation of the same chromatin region (11)(12)(13)(14). Here, we reveal that this CCNE1 proximal promoter region is targeted by another histone arginine methyl-transferase, the type I enzyme PRMT4 [coactivator-associated arginine methyltransferase (CARM1)] (19-25). PRMT4͞CARM1 was initially described as a transcriptional coactivator of the p160 family of nuclear receptor-associated factors (Src-1͞NCoA1, GRIP1͞TIF2͞Src-2͞ NC...

IntroductionRecent evidence suggests that tissue accumulation of senescent p16INK4a-positive cells during the life span would be deleterious for tissue functions and could be the consequence of inherent age-associated disorders. Osteoarthritis (OA) is characterized by the accumulation of chondrocytes expressing p16INK4a and markers of the senescence-associated secretory phenotype (SASP), including the matrix remodeling metalloproteases MMP1/MMP13 and pro-inflammatory cytokines interleukin-8 (IL-8) and IL-6. Here, we evaluated the role of p16INK4a in the OA-induced SASP and its regulation by microRNAs (miRs).MethodsWe used IL-1-beta-treated primary OA chondrocytes cultured in three-dimensional setting or mesenchymal stem cells differentiated into chondrocyte to follow p16INK4a expression. By transient transfection experiments and the use of knockout mice, we validate p16INK4a function in chondrocytes and its regulation by one miR identified by means of a genome-wide miR-array analysis.Resultsp16INK4a is induced upon IL-1-beta treatment and also during in vitro chondrogenesis. In the mouse model, Ink4a locus favors in vivo the proportion of terminally differentiated chondrocytes. When overexpressed in chondrocytes, p16INK4a is sufficient to induce the production of the two matrix remodeling enzymes, MMP1 and MMP13, thus linking senescence with OA pathogenesis and bone development. We identified miR-24 as a negative regulator of p16INK4a. Accordingly, p16INK4a expression increased while miR-24 level was repressed upon IL-1-beta addition, in OA cartilage and during in vitro terminal chondrogenesis.ConclusionsWe disclosed herein a new role of the senescence marker p16INK4a and its regulation by miR-24 during OA and terminal chondrogenesis.

Chromosome 17 is severely rearranged in breast cancer. Whereas the short arm undergoes frequent losses, the long arm harbors complex combinations of gains and losses. In this work we present a comprehensive study of quantitative anomalies at chromosome 17 by genomic arraycomparative genomic hybridization and of associated RNA expression changes by cDNA arrays. We built a genomic array covering the entire chromosome at an average density of 1 clone per 0.

Background: Both phenotypic and cytogenetic variability have been reported for clones of breast carcinoma cell lines but have not been comprehensively studied. Despite this, cell lines such as MCF-7 cells are extensively used as model systems.

Skeletal development and cartilage formation require stringent regulation of gene expression for mesenchymal stem cells (MSCs) to progress through stages of differentiation. Since microRNAs (miRNAs) regulate biological processes, the objective of the present study was to identify novel miRNAs involved in the modulation of chondrogenesis. We performed miRNA profiling and identify miR-29a as being one of the most down-regulated miRNAs during the chondrogenesis. Using chromatin immunoprecipitation, we showed that SOX9 down-regulates its transcription. Moreover, the over-expression of miR-29a strongly inhibited the expression of chondrocyte-specific markers during in vitro chondrogenic differentiation of MSCs. We identified FOXO3A as a direct target of miR-29a and showed a down- and up-regulation of FOXO3a protein levels after transfection of, respectively, premiR- and antagomiR-29a oligonucleotides. Finally, we showed that using the siRNA or premiR approach, chondrogenic differentiation was inhibited to a similar extent. Together, we demonstrate that the down-regulation of miR-29a, concomitantly with FOXO3A up-regulation, is essential for the differentiation of MSCs into chondrocytes and in vivo cartilage/bone formation. The delivery of miRNAs that modulate MSC chondrogenesis may be applicable for cartilage regeneration and deserves further investigation.

The aim of this study was to identify new microRNAs (miRNAs) that are modulated during the differentiation of mesenchymal stem cells (MSCs) toward chondrocytes. Using large scale miRNA arrays, we compared the expression of miRNAs in MSCs (day 0) and at early time points (day 0.5 and 3) after chondrogenesis induction. Transfection of premiRNA or antagomiRNA was performed on MSCs before chondrogenesis induction and expression of miRNAs and chondrocyte markers was evaluated at different time points during differentiation by RT-qPCR. Among miRNAs that were modulated during chondrogenesis, we identified miR-574-3p as an early up-regulated miRNA. We found that miR-574-3p up-regulation is mediated via direct binding of Sox9 to its promoter region and demonstrated by reporter assay that retinoid X receptor (RXR)α is one gene specifically targeted by the miRNA. In vitro transfection of MSCs with premiR-574-3p resulted in the inhibition of chondrogenesis demonstrating its role during the commitment of MSCs towards chondrocytes. In vivo, however, both up- and down-regulation of miR-574-3p expression inhibited differentiation toward cartilage and bone in a model of heterotopic ossification. In conclusion, we demonstrated that Sox9-dependent up-regulation of miR-574-3p results in RXRα down-regulation. Manipulating miR-574-3p levels both in vitro and in vivo inhibited chondrogenesis suggesting that miR-574-3p might be required for chondrocyte lineage maintenance but also that of MSC multipotency.

Chromosome 1 is involved in quantitative anomalies in 50 -60% of breast tumours. However, the structure of these anomalies and the identity of the affected genes remain to be determined. To characterise these anomalies and define their consequences on gene expression, we undertook a study combining array-CGH analysis and expression profiling using specialised arrays. Array-CGH data showed that 1p was predominantly involved in losses and 1q almost exclusively in gains. Noticeably, high magnitude amplification was infrequent. In an attempt to fine map regions of copy number changes, we defined 19 shortest regions of overlap (SROs) for gains (one at 1p and 18 at 1q) and of 20 SROs for losses (all at 1p). These SROs, whose sizes ranged from 170 kb to 3.2 Mb, represented the smallest genomic intervals possible based on the resolution of our array. The elevated incidence of gains at 1q, added to the wellestablished concordance between DNA copy increase and augmented RNA expression, made us focus on gene expression changes at this chromosomal arm. To identify candidate oncogenes, we studied the RNA expression profiles of 307 genes located at 1q using a home-made built cDNA array. We identified 30 candidate genes showing significant overexpression correlated to copy number increase. In order to substantiate their involvement, RNA expression levels of these candidate genes were measured by quantitative (Q)-RT -PCR in a panel of 25 breast cancer cell lines previously typed by array-CGH. Q -PCR showed that 11 genes were significantly overexpressed in the presence of a genomic gain in these cell lines, and 20 overexpressed when compared to normal breast.

In the human T cell receptor gamma (TRG) locus, fourteen variable (TRGV) genes belonging to four subgroups have been identified upstream of two constant region (TRGC) genes. Three joining segments, JP1, JP and J1, have been localized upstream of TRGC1, and two others, JP2 and J2, upstream of TRGC2. In this report, we demonstrate that a unique Xho I fragment of 120 kilobases (kb) contains the fourteen TRGV genes and that the hybridization of that fragment in pulsed-field gel electrophoresis (PFGE) allows linkage of the variable region to the constant region locus. We also show that the variable and the constant regions are remarkably close to each other since the distance between V11, the most 3' V gamma gene, and JP1, the most 5' J gamma segment, is only 16 kb. With its 14 V gamma genes, spanning 100 kb, the two C gamma genes and 5 joining segments covering less than 40 kb and only 16 kb separating the most 3' V gene from the most 5' J segment, the human TRG locus spans 160 kb of genomic DNA and represents a particularly condensed locus compared to the other rearranging gene loci.